Method for determination of presence or absence of peptide compound pyy3-36

ABSTRACT

The present invention provides a method for determining in a pharmaceutical test formulation the presence or absence of a peptide compound PYY 3-36  represented by the following amino acid sequence: H-Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp-Ala-Ser-Pro-Glu-Glu-Leu-Asn-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr-X (SEQ ID NO: 1), wherein X is OH or a carboxy acid-protecting group, the method comprising (1) preparing a solution by mixing the pharmaceutical test formulation with cucurbit[7]uril in a solvent; and (2) thermally analyzing the solution prepared in Step (1).

TECHNICAL FIELD

The present invention relates to a method for determining the presenceor absence of peptide compound PYY₃₋₃₆.

BACKGROUND ART

Recently, the harmful effects of obesity on health have become aproblem. Long-term or invasive treatment is required to promote andsustain weight loss. According to the U.S. Department of Health andHuman Services, approximately 300,000 deaths a year in the United Statesare associated with obesity, and the direct and indirect costsattributed to obesity exceed $100 billion.

Heretofore, anorectic agents such as sibutramine, phentermine, andorlistat have been commercially available; however, they haveunsatisfactory appetite-suppressing effects.

There has been growing expectation in recent years that natural peptidehormone, effective for regulating appetite, will be used in medicalapplications. Several pharmaceutical companies have started clinicaltrials and preclinical trials regarding the role of natural peptidehormone in obesity.

In particular, a peptide compound PYY₃₋₃₆ (hereinafter, sometimesabbreviated as “PYY₃₋₃₆”) represented by the following amino acidsequence:

H-Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp-Ala-Ser-Pro-Glu-Glu-Leu-Asn-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr-X,

wherein X is OH or a carboxy acid-protecting group, has been receivingattention as a natural peptide hormone. Intranasal administration ofPYY₃₋₃₆ has already been offered as a novel obesity treatment method.PYY₃₋₃₆ is included in secretions (hereinafter, sometimes referred to asa “secretion”) produced by endocrine cells of the human gut,particularly the stomach. The secretion is readily released based onhuman food intake, etc.

The secretion usually includes a peptide compound PYY₁₋₃₆ (hereinafter,sometimes abbreviated as “PYY₁₋₃₆”) represented by the following aminoacid sequence:

H-Tyr-Pre-Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp-Ala-Ser-Pro-Glu-Glu-Leu-Asn-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr-NH₂.

PYY₁₋₃₆ is a compound in which “Tyr-Pre-” is further bound to theN-terminal of PYY₃₋₃₆, and has a similar amino acid sequence to PYY₃₋₃₆.

When trypsin is present in the secretion, the trypsin may cleave PYY₁₋₃₆and PYY₃₋₃₆, which generates new peptide digests.

Pharmaceutical formulations containing PYY₃₋₃₆ can be made, for example,by extracting PYY₃₋₃₆ from the secretion. However, PYY₁₋₃₆, and peptidedigests derived from PYY₁₋₃₆ or PYY₃₋₃₆ may also be present in thepharmaceutical formulations during production.

When the pharmaceutical formulation containing PYY₃₋₃₆ is in solid form(tablets, powders, etc.), PYY₃₋₃₆ may be degraded due to long-termstorage, which results in the presence of peptide digests derived fromPYY₃₋₃₆ in the pharmaceutical formulation. It has been known that asolid peptide compound can be less stable than a compound in a solution(Non-patent Documents 1 to 4). In a solid peptide compound, a peptidebond is easily cleaved (Non-patent Document 5). Especially, when apeptide compound includes an arginine residue therein, the bond betweenthe arginine residue and the amino acid residue adjacent thereto iseasily breakable (Non-patent Document 6).

Therefore, in the PYY₃₋₃₆-containing pharmaceutical formulation, it isimportant to determine the presence or absence of PYY₃₋₃₆, i.e., thepresence or absence of peptide digests derived from PYY₃₋₃₆ duringproduction or after long-term storage. Additionally, when apharmaceutical formulation is made by extracting PYY₃₋₃₆ from asecretion, it is preferable to determine the presence or absence ofPYY₁₋₃₆ and peptide digests derived from PYY₁₋₃₆ during production.

Examples of the determination method include an HPLC method andelectrophoresis.

However, PYY₃₋₃₆, PYY₁₋₃₆, and the peptide digests thereof have a commonamino acid sequence from the C-terminal; therefore, it is difficult todetermine the presence or absence of PYY₃₋₃₆ using known methods.Further, the determination of the complete amino acid sequences of allpeptide compounds contained in a pharmaceutical formulation is both timeconsuming and complicated.

Non-patent Document 1:

Pearlman, R.; Nguyan, T. J., J. Pharm. Pharmacol. 1992, 44, 178

Non-patent Document 2:

Strickley, R. G.; Visor, G. C.; Lin, L.; Gu, L., Pharm. Res. 1989, 6,971

Non-patent Document 3:

Bhatt, N.; Patel, K.; Borchart, R. T., Pharm. Res. 1990, 7, 593

Non-patent Document 4:

Jordan, G. M.; Yoshioka, S.; Terao, T., J. Pharm. Pharmacol. 1994, 46,182

Non-patent Document 5:

Lai, M. C.; Topp, E. M., J. Pharm. Sci. 1999, 88, 489

Non-patent Document 6:

Kertscher, U.; Bienert, M.; Krause, E.; Sepetov, N. F.; Mehlis, B., Int.J. Pept. Protein Res. 1993, 41, 207

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

The main object of the present invention is to provide a method foreasily and accurately determining the presence or absence in apharmaceutical formulation of a peptide compound PYY₃₋₃₆ represented bythe following amino acid sequence:

H-Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp-Ala-Ser-Pro-Glu-Glu-Leu-Asn-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr-X,

wherein X is OH, or carboxy acid-protecting group.

MEANS FOR SOLVING THE PROBLEMS

In view of the problems of the foregoing prior art, the presentinventors conducted extensive research. As a result, they found that aspecific determination method can achieve the aforementioned object. Thepresent invention has been accomplished based on this finding.

The present invention relates to the following determination method.

Item 1

A method for determining in a pharmaceutical test formulation thepresence or absence of a peptide compound PYY₃₋₃₆ represented by thefollowing amino acid sequence:

H-Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp-Ala-Ser-Pro-Glu-Glu-Leu-Asn-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr-X,

wherein X is OH or a carboxy acid-protecting group, the methodcomprising:

(1) preparing a solution by mixing the pharmaceutical test formulationwith cucurbit[7]uril in a solvent; and (2) thermally analyzing thesolution prepared in Step (1).

Item 2

The determination method according to Item 1, wherein the presence orabsence of a peptide compound PYY₁₋₃₆ represented by the following aminoacid sequence:

H-Tyr-Pre-Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp-Ala-Ser-Pro-Glu-Glu-Leu-Asn-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr-NH₂,

and/or peptide digests generated by degradation of the peptide compoundPYY₁₋₃₆ is determined.

Item 3

The determination method according to Item 1, wherein peptide digestsgenerated by degradation of the peptide compound PYY₃₋₃₆ is detected.

The present invention relates to a method for determining in apharmaceutical formulation to be tested the presence or absence of apeptide compound PYY₃₋₃₆ represented by the following amino acidsequence:

H-Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp-Ala-Ser-Pro-Glu-Glu-Leu-Asn-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr-X,

wherein X is OH or a carboxy acid-protecting group, which comprises thesteps of (1) preparing a solution by mixing the pharmaceutical testformulation with a cucurbit[7]uril in a solvent; and (2) thermallyanalyzing the solution prepared in Step (1).

Examples of the carboxy acid-protecting group include NH₂, NH-R¹,NHCOR², OR³, SH, SR⁴, etc.

Examples of R¹ include an optionally substituted saturated hydrocarbongroup, an optionally substituted aryl group, an optionally substitutedheteroaryl group, an optionally substituted aralkyl group, etc.

Optionally substituted saturated hydrocarbon groups are not particularlylimited. Specific examples thereof include C₁₋₂₀ linear or branchedalkyl groups and C₃₋₁₂ cycloalkyl groups. Specific examples of C₁₋₂₀linear or branched alkyl groups include methyl, ethyl, propyl,isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl,heptyl, octyl, nonyl, decyl, undecyl, dodecyl, icosyl, etc. Specificexamples of C₃₋₁₂ cycloalkyl groups include cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, cyclododecyl, etc.

Substitute groups of optionally substituted saturated hydrocarbon groupsare not particularly limited. Specific examples thereof include alkoxy,aryloxy, siloxy, dialkylamino, etc. Examples of alkoxy groups includeC₁₋₆ alkoxy groups. Specific examples include methoxy, ethoxy, hexyloxy,etc. Examples of aryloxy groups include C₆₋₁₂ aryloxy groups, such asphenoxy, naphthyloxy, etc. Examples of siloxy groups includetrimethylsiloxy, triethylsiloxy, triisopropylsiloxy,tert-butyldimethylsiloxy, etc. Examples of dialkylamino groups includedimethylamino, diethylamino, etc.

The substitution site and the number of substituents of optionallysubstituted saturated hydrocarbon groups are not particularly limited sofar as the effect of the invention is not impaired.

Optionally substituted aryl groups are not particularly limited, andinclude C₆₋₁₄ aryl. Specific examples thereof include phenyl,1-naphthyl, 2-naphthyl, biphenylyl, anthryl, etc.

Substitute groups of optionally substituted aryl groups are not limitedso far as the effect of the invention is not impaired. Specific examplesthereof include C₁₋₆ alkyl, C₆₋₁₄ aryl, 5 to 10-membered aromaticheterocycle, alkoxy, aryloxy, siloxy, dialkylamino, etc.

Examples of C₁₋₆ alkyl groups include methyl, ethyl, propyl, isopropyl,butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, etc.

Examples of C₆₋₁₄ aryl groups include phenyl, 1-naphthyl, 2-naphthyl,biphenylyl, 2-anthryl, etc.

Examples of 5 to 10-membered aromatic heterocycles include 2-, or3-thienyl; 2-, 3-, or 4-pyridyl; 2-, 3-, 4-, 5-, or 8-quinolyl; 1-, 3-,4-, or 5-isoquinolyl; 1-, 2-, or 3-indolyl; 2-benzothiazolyl;2-benzo[b]thienyl; benzo[b]furanyl; etc.

Examples of alkoxy groups include C₁₋₆ alkoxy groups, such as methoxy,ethoxy, hexyloxy, etc.

Examples of aryloxy groups include C₆₋₁₂ aryloxy groups, such asphenoxy, naphthyloxy, etc.

Examples of siloxy groups include trimethylsiloxy, triethylsiloxy,triisopropylsiloxy, tert-butyldimethylsiloxy, etc.

Examples of dialkylamino groups include dimethylamino, diethylamino,etc.

The substitution site and the number of substituents of optionallysubstituted aryl groups are not particularly limited sofar as the effectof the invention is not impaired.

Optionally substituted heteroaryl groups are not particularly limited.Examples thereof include optionally condensed 5 to 14-membered aromaticheterocycles with 1 to 3 atoms selected from sulfur atoms, oxygen atoms,and nitrogen atoms. Specific examples include furil, thienyl, pyrrolyl,pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, isothiazolyl, thiazolyl,1,2,3-oxadiazolyl, triazolyl, tetrazolyl, thiadiazolyl, pyridyl,pyridazinyl, pyrimidinyl, pyrazinyl, indolyl, indazolyl, purinyl,quinolyl, isoquinolyl, phthalazinyl, naphthyridinyl, quinoxalinyl,quinazolinyl, cynolinyl, pteridinyl, carbazolyl, carbonyl,phenanthridinyl, acridinyl, etc.

The type, substitution site, and number of substituents of optionallysubstituted heteroaryl groups are the same as those described in thedescription of the aryl groups.

Examples of optionally substituted aralkyl groups include benzyl,phenethyl, phenylpropyl, etc. The type, substitution site, and number ofsubstituents of optionally substituted aralkyl groups are the same asthose described in the description of the aryl group.

Examples of R², R³, and R⁴ are the same as those of R¹ above.

Methyl and ethyl groups are preferably used as R¹, R², R³, and R⁴. WhenR¹, R², R³, and R⁴ are methyl or ethyl groups, PYY₃₋₃₆ can be suitablydissolved in water, which is used as a solvent, during the production ofthe solution described later.

In particular, X is preferably NH₂ in the determination method of thepresent invention.

The present invention relates to a method for determining the presenceor absence of PYY₃₋₃₆ in a pharmaceutical test formulation. PYY₃₋₃₆ canbe extracted from a secretion produced by endocrine cells of the humangut, particularly the stomach. When the pharmaceutical test formulationis made using the obtained secretion, PYY₁₋₃₆ and digests thereof mayalso be present in the pharmaceutical formulation. When a pharmaceuticalformulation containing PYY₃₋₃₆ is stored for a long period of time,PYY₃₋₃₆ may be degraded to generate peptide digests. Particularly, thebond between arginine and tyrosine inside PYY₁₋₃₆ and PYY₃₋₃₆ isrelatively weaker than the bond between other amino acids, and is moreeasily broken. Accordingly, PYY₁₋₃₆ is degraded (the bond betweenarginine and tyrosine is broken), which allows for easy generation ofpeptide digests having a tyrosine residue at the N-terminal. Further,the degradation of PYY₃₋₃₆ (the bond between arginine and tyrosine isbroken) allows for easy generation of peptide digests having a tyrosineresidue at the N-terminal and peptide digests having an isoleucineresidue at the N-terminal.

PYY₁₋₃₆, peptide digests derived from PYY₁₋₃₆, and peptide digestsderived from PYY₃₋₃₆, each having a tyrosine residue at the N-terminal,are reacted (forming a complex) with cucurbit[7]uril (hereinafter,sometimes referred to as “CB[7]”) to generate heat. According to thedetermination method of the present invention, the presence or absenceof PYY₃₋₃₆ can be determined by detecting PYY₃₋₃₆-derived peptidedigests having a tyrosine residue at the N-terminal through the thermalanalysis of the mixture obtained by mixing the pharmaceutical testformulation and CB[7] in a solution. Further, the purity of PYY₃₋₃₆ inthe pharmaceutical test formulation can be measured by detecting PYY₁₋₃₆and PYY₁₋₃₆-derived peptide digests both having a tyrosine residue atthe N-terminal.

Step (1)

In Step (1), the pharmaceutical test formulation is mixed withcucurbit[7]uril in a solvent to prepare a solution.

Pharmaceutical Test Formulation

According to the determination method of the invention, the presence orabsence of PYY₃₋₃₆ in the pharmaceutical test formulation can bedetermined. That is, the presence or absence of PYY₃₋₃₆ (the presence orabsence of PYY₃₋₃₆ degradation) can be determined by detecting peptidedigests that are generated as a result of PYY₃₋₃₆ degradation.

Examples of peptide digests generated as a result of PYY₃₋₃₆ degradationinclude those having a tyrosine residue at the N-terminal, those havingan isoleucine residue at the N-terminal, etc.

Examples of PYY₃₋₃₆-derived peptide digests having a tyrosine residue atthe N-terminal (hereinafter sometimes abbreviated as “Digest A”) includeTyr-X, Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr-X,Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr-X,Tyr-Tyr-Ala-Ser-Leu-Arg-His,Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg,Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg, etc. These may be used singly or ina combination of two or more in a pharmaceutical test formulation.

Examples of PYY₃₋₃₆-derived peptide digests having an isoleucine residueat the N-terminal (hereinafter sometimes abbreviated as “Digest B”)includeH-Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp-Ala-Ser-Pro-Glu-Glu-Leu-Asn-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg,H-Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp-Ala-Ser-Pro-Glu-Glu-Leu-Asn-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His,H-Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp-Ala-Ser-Pro-Glu-Glu-Leu-Asn-Arg.These may be used singly or in a combination of two or more in apharmaceutical test formulation.

According to the determination method of the present invention, thepresence or absence of peptide compound PYY₁₋₃₆ represented by thefollowing amino acid sequence:

H-Tyr-Pre-Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp-Ala-Ser-Pro-Glu-Glu-Leu-Asn-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr-NH₂,

and/or peptide digests that are generated by the degradation of peptidecompound PYY₁₋₃₆ can be determined in a pharmaceutical test formulation.

Examples of peptide digests generated by the degradation of PYY₁₋₃₆include those having a tyrosine residue at the N-terminal.

Examples of PYY₁₋₃₆-derived peptide digests having a tyrosine residue atthe N-terminal (hereinafter sometimes abbreviated as “Digest C”) includeTyr-NH₂, Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr-NH₂,Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr-NH₂,Tyr-Tyr-Ala-Ser-Leu-Arg-His,Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg,Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg,H-Tyr-Pre-Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp-Ala-Ser-Pro-Glu-Glu-Leu-Asn-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg,H-Tyr-Pre-Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp-Ala-Ser-Pro-Glu-Glu-Leu-Asn-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His,H-Tyr-Pre-Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp-Ala-Ser-Pro-Glu-Glu-Leu-Asn-Arg,and the like. These may be used singly or in a combination of two ormore in a pharmaceutical test formulation.

The amounts of PYY_(3-36,) Digest A, Digest B, PYY₁₋₃₆, and Digest C ina pharmaceutical test formulation are not particularly limited.According to the determination method of the invention, when at leastone member selected from the group consisting of Digest A having atyrosine residue at the N-terminal, PYY₁₋₃₆, and Digest C is containedin an amount 1 wt % or more in total, Digest A or the like can besuitably detected, which makes it possible to determine the presence orabsence of PYY₃₋₃₆ with high accuracy.

The pharmaceutical test formulation may include the following knowncomponents, which are usually contained in pharmaceutical formulations.For example, peptide compounds other than PYY₃₋₃₆, Digest A, Digest B,PYY₁₋₃₆, and Digest C may be contained.

The forms of pharmaceutical formulations are not particularly limited.Typical examples include tablets, pills, powders, solutions,suspensions, emulsions, granules, capsules, suppositories, injections(solutions, suspensions, etc.) and the like. In the examination methodof the invention, pharmaceutical formulations in such forms can be usedsingly or in a combination of two or more.

CB[7]

CB[7] is a barrel-shaped molecule, which is shown in FIG. 1.

The presence or absence of PYY₃₋₃₆ cannot be determined with highaccuracy when cucurbiturils (e.g., cucurbit[6]uril, cucurbit[8]uril)other than cucurbit[7]uril are used.

Solution Preparation

The solution is prepared by mixing the pharmaceutical test formulationwith CB[7] in a solvent.

When the pharmaceutical test formulation includes Digest A, PYY_(1-36,)Digest C, etc., mixing the pharmaceutical test formulation with CB[7] ina solution causes at least one peptide compound selected from Digest A,PYY_(1-36,) and Digest C to react with CB[7] (forming a complex); andgenerates heat.

Usable solvents are not particularly limited, but the solvent ispreferably water. CB[7] can be suitably dissolved in water.

The concentration of the pharmaceutical test formulation in the solutionis not particularly limited, but is preferably 0.01 to 1 mmol/l, morepreferably 0.02 to 0.5 mmol/l, and most preferably 0.02 to 0.05 mmol/l.

The concentration of CB[7] in the solution is not particularly limited,but is preferably 0.1 to 10 mmol/l, more preferably 0.2 to 5 mmol/l, andmost preferably 0.2 to 0.5 mmol/l.

The pH of the solution is not limited, but preferably 1 to 8, morepreferably 3 to 8, and most preferably 5 to 7. In particular, thedegradation of PYY₃₋₃₆, PYY₁₋₃₆ or the like can be further prevented ata pH of 3 or more.

In the determination method of the present invention, the solution mayinclude salt, but preferably not. Salt often prevents a reaction(complex formation) of CB[7] with Digest A or the like.

Examples of salts include neutral salts that are dissolved in a solutionof Na₂SO₄, K₂SO₄, MgSO₄, NaCl, KCl, MgCl₂, NaNO₃, KNO₃, Mg(NO₃)₂,Ca(NO₃)₂, Na₃PO₄, K₃PO₄, etc; slightly acidic salts that are dissolvedin a solution of NaH₂PO₄, KH₂PO₄, etc; slightly basic salts that aredissolved in a solution of Na₂CO₃, NaHCO₃, K₂CO₃, KHCO₃, Na₂HPO₄,K₂HPO₄, CH₃COONa, and CH₃COOK, etc. Such salts can be used singly or ina combination of two or more.

The concentration of salt in a solution is generally 0.5 mol/l or less,preferably 0.1 mol/l or less, more preferably 10 mmol/l or less, andmost preferably 0 to 1 mmol/l. When the concentration of salt in asolution is more than 0.5 mol/l, a reaction (complex formation) of CB[7]with Digest A or the like is difficult to proceed. When theconcentration of salt in a solution is more than 10 mmol/l, a reaction(complex formation) between CB[7] and Digest A or the like suitablyadvances, which makes it possible to determine the presence or absenceof PYY₃₋₃₆ with high accuracy.

The mixing method is not particularly limited. For example, the thermalanalysis in Step (2) is conducted using an isothermal titrationcalorimeter in the following manner. The solution that is formed by thedissolution of CB[7] and salt in a solvent (hereinafter sometimesreferred to as “Solution B”) is added dropwise by a specified amount(e.g., 0.01 ml) to the solution formed by the dissolution of thepharmaceutical formulation in the solvent (hereinafter sometimesreferred to as “Solution A”) under thermal balance.

The concentration of the pharmaceutical test formulation in Solution Ais not limited, but is preferably 0.01 to 1 mmol/l, more preferably 0.02to 0.5 mmol/l, and most preferably 0.02 to 0.05 mmol/l. Theconcentration of CB[7] in Solution B is not limited, but is preferably0.1 to 10 mmol/l, more preferably 0.2 to 5 mmol/l, and most preferably0.2 to 0.5 mmol/l.

The concentration of salt in Solution B is not limited, but ispreferably 10 mmol/1 or less, and more preferably 0 to 2 mmol/l.

Step (2) will be described in detail below with reference to examples inwhich the determination method of the invention is performed using anisothermal titration calorimeter.

Step (2)

In Step (2), the solution obtained in Step (1) is thermally-analyzed.Specifically, the amount of heat is quantified each time Solution B isadded dropwise. The known calorimeters may be used for thermal analysisof the solution. In the present invention, it is particularly preferableto use an isothermal titration calorimeter for measurement of heat.

In the determination method of the invention, heat generated during themixing of pure PYY₃₋₃₆ and CB[7] in a solution is quantified beforesolution preparation in Step (1). Namely, a calibration curve isprepared in advance.

A PYY₃₋₃₆-containing solution and Solution B are first prepared, andSolution B may be added dropwise to the PYY₃₋₃₆-containing solution by acertain amount (e.g., 0.01 ml per each).

The PYY₃₋₃₆-containing solution can be prepared by dissolving PYY₃₋₃₆ inthe above-mentioned solvent.

The concentration of PYY₃₋₃₆ in the PYY₃₋₃₆-containing solution is notlimited as long as the concentration is approximately equivalent to thatof Solution A, and preferably 0.01 to 1 mmol/l, more preferably 0.02 to0.5 mmol/l, and most preferably 0.02 to 0.05 mmol/l.

The calibration curve can be prepared by plotting the molar ratio ofPYY₃₋₃₆ and the added CB[7] on the abscissa and the amount of heatgenerated at each drop on the ordinate.

The presence or absence of PYY₃₋₃₆ in the pharmaceutical testformulation can be determined by comparing the plot of the amounts ofheat on the calibration curve and that obtained through the heatanalysis of the solution prepared in Step (1). Specifically, it ispossible to determine the presence or absence of PYY₁₋₃₆, Digest C, orthe like during production; and it is possible to determine if PYY₃₋₃₆is not degraded but effectively present in the long-storedpharmaceutical formulation.

EFFECTS OF THE INVENTION

According to the determination method of the invention, the presence orabsence of peptide compound PYY₃₋₃₆ represented by the following aminoacid sequence:

H-Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp-Ala-Ser-Pro-Glu-Glu-Leu-Asn-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr-X,

wherein X is OH, or a carboxy acid-protecting group that can be easilyand accurately (range of error within approximately 1 to 2%) determinedin a pharmaceutical formulation. Specifically, according to thedetermination method of the invention, the presence or absence ofPYY₃₋₃₆ in a long-stored pharmaceutical formulation can be determined bydetecting peptide digests generated by peptide compound PYY₃₋₃₆degradation. Further, according to the determination method of theinvention, the presence or absence of PYY₁₋₃₆ and the digests thereofcan be determined by detecting PYY₁₋₃₆ and/or peptide digests that aregenerated by peptide compound PYY₁₋₃₆ degradation. Accordingly, thepresence or absence of PYY₁₋₃₆ and the digests thereof, which may bepresent in the pharmaceutical formulation during production, can beeasily and accurately (range of error within approximately 1 to 2%)determined by the determination method of the present invention.

Accordingly, the determination method of the invention is very effectivemeans of qualitatively analyzing a PYY₃₋₃₆-containing pharmaceuticalformulation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a chemical structure of cucurbit[7]uril.

FIG. 2 is a graph in which the heat content (▪) obtained in ReferenceExample 1 and the heat content (▾) obtained in the “Formation ofCalibration Curve” in Example 1 are plotted.

FIG. 3 is a graph in which the heat content (O) obtained in the“Formation of Calibration Curve” and the heat content (▪) obtained inthe “Determination of the Presence or Absence of PYY₃₋₃₆” in Example 1are plotted.

FIG. 4 is a graph in which the heat content (▪) obtained in the“Formation of Calibration Curve” and the heat content (O) obtained inthe “Determination of the Presence or Absence of PYY₃₋₃₆” in Example 2are plotted.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is described in further detail with reference tothe following Reference Examples and Examples. However, the presentinvention is not limited thereto.

REFERENCE EXAMPLE 1

A 0.03 mmol/l Tyr-NH₂ solution (1.5 ml ) was loaded into a reaction cellof an ultra-sensitive isothermal titration calorimeter VP-ITC (producedby MicroCal Inc.)

The colorimeter syringe was filled with a 0.3 mmol/l CB[7]-containingsolution (0.25 ml).

The calorimeter was set in the state of thermal equilibrium, and 0.01 mlof CB[7]-containing solution was added to a reaction cell a total of 25times. The resulting data was automatically integrated using ORIGIN 7.0(produced by MicroCal Inc.) The results are shown in FIG. 2 (▾).

FIG. 1. reveals that the peptide compound having a tyrosine residue atthe N-terminal is reacted with CB[7] (forming a complex) to generateheat.

EXAMPLE 1 Formation of Calibration Curve

A solution (1.5 ml, concentration: 0.03 mmol/l ) of PYY₃₋₃₆ representedby the following amino acid sequence:

H-Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp-Ala-Ser-Pro-Glu-Glu-Leu-Asn-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr-NH₂

was loaded into a reaction cell of an ultra-sensitive isothermaltitration calorimeter VP-ITC (produced by MicroCal Inc.)

The colorimeter syringe was filled with a CB[7]-containing solution(0.25 ml, 0.3 mmol/l ).

The calorimeter was set in the state of thermal equilibrium, and 0.01 mlof CB[7]-containing solution was added to a reaction cell 25 times intotal. The resulting data was automatically integrated using ORIGIN 7.0(produced by MicroCal Inc.) The results are shown in FIG. 2 (▪).

Determination of the Presence or Absence of PYY₃₋₃₆

Subsequently, the pharmaceutical test formulation containing PYY₃₋₃₆ andTyr-NH₂ (the molar ratio of PYY₃₋₃₆ to Tyr-NH₂ is 95:5) was dissolved inwater to thereby prepare a 0.03 mmol/l pharmaceutical testformulation-containing solution. The resulting solution was loaded intoa reaction cell of the calorimeter. Further, the colorimeter syringe wasfilled with 0.25 ml of CB[7]-containing solution.

The heat content was measured using the same method described above, andthe resulting data was automatically integrated. The results (▪) areshown in FIG. 3. The calibration curve (O) is also shown in FIG. 3 forcomparison.

FIG. 3 reveals that the mixture of the pharmaceutical test formulation(the molar ratio of PYY₃₋₃₆ and Tyr-NH₂ is 95:5) generates more heatthan that of pure PYY₃₋₃₆. Specifically, FIG. 3 reveals that, comparedto pure PYY₃₋₃₆, the pharmaceutical test formulation includes Tyr-NH₂ tocompensate for the shortage of PYY₃₋₃₆, which results in the differencein the heat quantity.

EXAMPLE 2 Formation of Calibration Curve

A calibration curve was prepared in the same manner as in Example 1. Theresults (▪) are shown in FIG. 4.

Determination of the Presence or Absence of PYY₃₋₃₆ (PYY₁₋₃₆)

Subsequently, the pharmaceutical test formulation containing PYY₃₋₃₆ andPYY₁₋₃₆ (the molar ratio of PYY₃₋₃₆ to PYY₁₋₃₆ is 95:5) was dissolved inwater to thereby prepare a 0.03 mmol/l pharmaceutical testformulation-containing solution. The resulting solution was loaded intoa reaction cell of the calorimeter. Further, the colorimeter syringe wasfilled with 0.25 ml of CB[7]-containing solution.

The heat content was measured using the same method described above, andthe resulting data was automatically integrated. FIG. 4 shows theresults (O).

FIG. 4 reveals that the mixture of the pharmaceutical test formulation(the molar ratio of PYY₃₋₃₆ and PYY₁₋₃₆ is 95:5) generates more heatthan that of pure PYY₃₋₃₆. Specifically, FIG. 4 reveals that, comparedto pure PYY₃₋₃₆, the pharmaceutical test formulation includes PYY₁₋₃₆ tocompensate for the shortage of PYY₃₋₃₆, which results in the differencein the heat quantity.

1. A method for determining in a pharmaceutical test formulation thepresence or absence of a peptide compound PYY₃₋₃₆ represented by thefollowing amino acid sequence:H-Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp-Ala-Ser-Pro-Glu-Glu-Leu-Asn-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr-X  (SEQ ID NO: 1), wherein X is OH or a carboxy acid-protecting group,the method comprising: (1) preparing a solution by mixing thepharmaceutical test formulation with cucurbit[7]uril in a solvent; and(2) thermally analyzing the solution prepared in Step (1).
 2. Thedetermination method according to claim 1, wherein the presence orabsence of a peptide compound PYY₁₋₃₆ represented by the following aminoacid sequence:H-Tyr-[[Pre]]Pro-Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp-Ala-Ser-Pro-Glu-Glu-Leu-Asn-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr-NH₂  (SEQ ID NO: 2), and/or peptide digests generated by degradation of thepeptide compound PYY₁₋₃₆ is determined.
 3. The determination methodaccording to claim 1, wherein peptide digests generated by degradationof the peptide compound PYY₃₋₃₆ is detected.